The lack of efficient gene delivery systems is the most important barrier to the use of gene therapy for cancer. There is an urgent need to develop vectors that are capable of selective, efficient, and safe delivery of genes to tumors. Until this need is met, the paradigm shift in cancer treatment that gene therapy represents will remain unrealized. It is thus the long-term goal of this research program to improve the efficacy of cancer treatment by facilitating the development of a new generation of improved vectors for cancer gene therapy. Toward this end, the objective of this proposal is to develop a gene-delivery strategy that will selectively target tumor cells while concomitantly overcoming the anti-vector immunity of the host and reducing the level of undesired transduction of normal tissues. The central hypothesis to be tested is that this objective can be accomplished through sequential use of immunologically distinct adenovirus (Ad) vectors targeted to tumors using a versatile strategy of tropism modification. The rationale for this study is that overcoming the vector immunogenicity and specificity problems will be a major vertical step toward making cancer gene therapy a clinical reality. The central hypothesis will be tested and thereby the objective of this project will be achieved by realizing two specific aims: Specific Aim 1. Develop a panel of antigenically distinct tumor-targeted Ad vectors. Specific Aim 2. Test the ability of the designed vectors to repeatedly target tumors in the presence of anti-Ad immunity. In this project, a diverse panel of receptor- binding Ad fiber proteins that represent the natural diversity of these molecules will be genetically modified to target a major molecular marker on human tumors, the Her2 receptor. These proteins will be made Her2- specific through the use of novel, rationally designed protein ligands, affibodies, whose size, biosynthesis, stability, and high affinity make them uniquely suited for Ad vector targeting. To make these fiber-affibody chimeras, we will use a novel targeting strategy based on the evolutionary conservation of the Ad fiber structure and thus applicable to a variety of Ad serotypes. The key structural and functional features of the designed fibers will be tested to confirm their suitability for Ad targeting. Next, a panel of Her2-targeted Ad vectors containing these designed proteins will be generated and tested in vitro using Her2-expressing tumor cells. Last, through the use of these new vectors in a transgenic animal model of breast cancer, we will demonstrate the feasibility of repeated gene delivery to target tumors despite preexisting anti-Ad immunity. This first demonstration of this key feasibility of gene therapy paradigm in our study will be of major significance because it will open the door to the development of efficacious gene interventions in humans, thereby facilitating the translation of this technology into clinical use. The success of the proposed work is supported by our preliminary findings, the collective expertise of our research team, and the exceptional research environment at The University of Texas M. D. Anderson Cancer Center. PUBLIC HEALTH RELEVANCE: The proposed research is directly relevant to public health because it seeks to develop a new genetic strategy of treating and imaging human tumors. Accomplishing this goal will make it possible for the first time to repeatedly and efficiently deliver gene therapeutic and diagnostic agents to target tumors in cancer patients, most of who cannot benefit from genetic interventions at present because of the lack of appropriate gene delivery vehicles.